The neural crest is a multipotent embryonic cell population that contributes to diverse derivatives, including peripheral ganglia, cartilage and bone of the face, and melanocytes. We have proposed and tested a multistep gene regulatory network (GRN), comprised of a logical series of distinct regulatory steps that act in concert to imbue the cranial neural crest its defining traits. However, there are significant differences in developmental potential and migratory pathways of different neural crest populations arising at different axial levels. Here, we propose to explore GRN differences along the neural axis, focusing on premigratory neural crest cells from two distinct regions: cranial versus trunk. Our preliminary transcriptome analysis reveals many transcription factors and signaling molecules specific to the cranial but not trunk neural crest or vice versa. Our goal is to determine the position of these genes in the cranial versus trunk GRNs. This systems level strategy will provide understanding of why NC GRNs produces a particular regulatory state for use in preprogramming these cells to a different state. The aims are: Aim 1: Multiplex perturbation analysis of GRN connections at cranial and trunk levels. With the genomewide representation ofthe active transcriptome of premigratory cranial and trunk neural crest in hand, we will perform loss-of-function experiments to perturb gene function and quantitate subsequent global transcriptional changes in putative target genes in single embryos using Nanostring analysis. Aim 2: Phylogenomic and funcfional analysis/dissection of neural crest enhancers. We will identify cisregulatory elements that mediate expression of key GRN factors in cranial versus trunk neural crest populations. We will perform multidimensional modeling that incorporates results of transcriptome data and active enhancers with functional perturbation results into representational models of neural crest GRNs. Aim 3: Reengineering ofthe trunk neural crest program to test skeletogenic potenfial. Using GRN informafion, we will challenge the fate of trunk NC by reengineering their regulatory circuits and observing if misexpression/deletion of key GRN subcircuits affects their identify and ability to contribute to cartilage.